Assay for camp based on exclusive binding of camp to the b-binding sites of camp dependent protein kinase (capk)

ABSTRACT

The present invention provides a method for assaying for cAMP in a sample, said method comprising contacting a sample with an unknown cAMP content with a polypeptidic cAMP binding agent and optionally with a labelled cAMP and detecting conjugates of cAMP or labelled cAMP and said binding agent, characterized in that said binding agent comprises functional cAPK cAMP B-binding sites only.

This invention relates to a method for assaying for cyclic adenosinemonophosphate (cAMP) and other cyclic nucleotides and cyclic nucleotideanalogs and to kits for use in such methods.

cAMP or adenosine 3′,5′-cyclic monophosphate comprises a ribofuranosemonosaccharide unit with a purine group attached at the 1′ position anda phosphate group attached at the 3′ and 5′ positions. cAMP is formed bycyclization of ATP and is a “second messenger” which functions as amediator of hormone action for a range of hormones. In particular itserves to activate key enzymes known as protein kinases.

cAMP is widely distributed in the body and has been assayed for inconnection with a broad range of conditions, eg thyroid malfunction,calcium metabolism disorders, etc.

Several assays for cAMP are available commercially, especially fromAmersham plc, UK. The most widely used assays are based on competitionbetween cAMP and a labelled (eg radiolabelled) cAMP or cAMP analog for ahigh affinity binding site either on an immunoglobulin or on a naturallyoccurring cAMP binding protein, in particular cAMP dependent proteinkinase isoenzyme type I (cAPKI).

While such assays have been available for some decades now, they arenonetheless less accurate than is desirable and the antibody basedassays are unduly sensitive to the agents (eg EDTA) that are added tocAMP containing samples to prevent degradation of cAMP before the assaycan be carried out.

There is thus a need for an improved assay for cAMP.

We have now found that such an improved assay can be achieved by usingas a cAMP binding agent a polypeptide comprising in functional form onlyone type of cAMP binding site of a cAMP dependent protein kinase (cAPK).cAPK, which exists as type I (cAPKI) and type II (cAPKII), has two typesof cAMP binding sites, the A and B sites, on each of the regulatory (R)subunits located respectively towards and at the C-terminus. Thus forcAPKI, which is a dimer, there are four binding sites Aα, Aβ, Bα and Bβ.In terms of amino acid sequence the A sites on the α and β chains areidentical as are the B sites on the α and β chains.

cAMP dependent protein kinases are discussed for example in Taylor etal. Ann. Rev. Biochem. 59: 971 (1990), Doskeland et al. Biochim.Biophys. Acta 1178: 249-258 (1993), Francis et al. Ann. Rev. Physiol.56: 237-272 (1994) and Johnson et al. Chem. Rev. 101: 2243-2270 (2001).The A and B sites of the polypeptide RIα (the regulatory (R) subunit ofthe a chain of cAMP dependent protein kinase type I) is described by Yuet al. in Science 269: 807-813 (1995) who co-crystallized cAMP and atruncated RIα with intact A and B sites. RI has two forms, RIα and RIβwhich are very similar in terms of amino acid sequence in the cAMPbinding domains. The R subunits of cAMP dependent protein kinase type II(i.e. RIIα and RIIβ) have A and B sites that are similar to thecorresponding sites in RIα. (See Diller et al. Structure 9: 73-82(2001)).

In mammalian R subunits, the A site comprises roughly amino acidresidues 143-260 (RIα) or 158-277 (RIIβ) and the B site comprisesroughly residue 260-374 (RIα) or 277-426 (RIIβ).

Thus viewed from one aspect the invention provides a method for assayingfor cAMP in a sample, said method comprising contacting a sample with anunknown cAMP content with a polypeptidic cAMP binding agent andoptionally with a labelled cAMP, and detecting conjugates of cAMP orlabelled cAMP and said binding agent, characterized in that said bindingagent comprises functional cAPK cAMP B-binding sites only.

The functional site is a B site, more preferably a RI B-site, mostpreferably a RIα B-site.

By “functional site” is meant herein that the binding site is capable ofbinding cAMP with a K_(D) of no more than 300% of that of the site innative human cAPK, preferably no more than 200%, more preferably no morethan 150% and especially no more than 110% . (The lower the K_(D), thehigher the binding affinity).

Besides the functional cAMP binding site (or sites), the binding agentmay contain non-functional cAMP binding sites, e.g. of a different type.By a non-functional site is meant a site corresponding to a native cAMPbinding site in some or all of its amino acid sequence but which has aK_(D) for cAMP binding which is more than 300% of that of thecorresponding site in native human cAPK, preferably more than 500% andmore preferably more than 10000%.

By a “cAPK cAMP binding site” is meant a polypeptide sequence having theability to bind cAMP and having a sequence homology with a nativemammalian cAPK cAMP binding site sequence of at least 60%, preferably atleast 80%, most preferably at least 95%. The cAPK cAMP binding site inthe binding agent used in the assay method of the invention thus neednot be identical to a native mammalian cAPK cAMP binding site, e.g. dueto insertions, deletions, substitutions or transpositions of one or moreamino acids. The cAPK cAMP binding site in the binding agent willpreferably contain at least 95% of the amino acid sequence of thecorresponding cAPK cAMP binding site common to human and bovine cAPK,i.e. the highly conserved section of the sequence. The term “functionalcAPK cAMP binding site” is also considered to extend to cAMP bindingpolypeptide sequences having a functional topology equivalent to nativecAPK cAMP binding sites, eg synthetic polypeptide sequences havingsimilar tertiary structure presenting the same or equivalent functionalgroups within the cAMP binding pocket.

As is routine in diagnostic assays, the detection of the analyte:bindingagent conjugate may be direct or indirect. Thus for example wherelabelled cAMP is used in the assay method a signal from the label in thelabelled cAMP:binding agent conjugate, for example a radiation emission,may be detected directly and the content of cAMP in the sample inferredfrom this. Alternatively the signal from the label in the uncomplexedlabelled cAMP may be detected directly and the content of cAMP in thesample again inferred from this. As a further alternative, a secondarybinding agent which binds to cAMP:primary binding agent conjugates or touncomplexed primary binding agent and which gives rise to a directlydetectable signal or event may be used and from the detected signal thecontent of cAMP in the sample may again be inferred. All such systems ofanalyte detection are encompassed by the assay method of the invention.

Viewed from a further aspect the invention also provides a kit for acAMP assay, said kit comprising a polypeptidic primary binding agentcapable of binding cAMP; optionally, a labelled cAMP; and optionally asecondary binding agent; characterized in that said primary bindingagent comprises functional cAPK cAMP B-binding sites only. The kit ofthe invention also preferably includes instructions for the performanceof the assay method.

Viewed from a still further aspect the invention provides a polypeptidiccAMP binding agent which comprises functional cAPK cAMP B-binding sitesonly, and compositions and items comprising said binding agent.

By compositions containing the binding agent are included liquid,semi-solid (e.g. gel) and solid (e.g. pulverulent) compositions,typically including further substances such as liquid or solid carriersor gel forming agents, preservatives, pH modifiers, etc. By itemscomprising the binding agent are included for example solid orsemi-solid structures coated or impregnated with the binding agent, e.g.beads, plates, tubes, membranes, fibres, etc with the binding agentimmobilized thereon.

The cAMP binding agent used according to the invention is preferably arecombinant protein comprising one or more, e.g. 1, 2 or 3, cAPK cAMPbinding sites, especially preferably bovine cAPK cAMP binding sites,optionally a fusion protein comprising a section couplable to asubstrate surface (a “surface binding region”). Where the binding agentcontains two or more cAPK cAMP binding sites, these should be of thesame type, thus for example both or all may be RIα B-sites. The proteinmay, but need not necessarily, include a disabled cAPK cAMP bindingsite. By disabled in this context is meant that the amino acid sequenceof the cAMP binding site is modified to substantially reduce oreliminate the cAMP binding ability of the site, e.g. by substitution,insertion or deletion of one or more amino acids so as to change thetertiary structure or the functional topology of the site. In apreferred embodiment, the binding agent thus has one functional RIB-site and one disabled RI A-site. A-site disablement is convenientlyachieved by mutagenesis replacing a highly conserved Gly residue(depending on the species this may be residue 199, 200 or 201) inmammalian, e.g. human or bovine, RIα by Glu. This can be done using PCRand a standard mutagenesis kit (e.g. Strategene Quick Change). Theefficacy of A-site disablement can be verified by equilibrium binding,e.g. using the method of Hummel et al. BBA 63: 530-532 (1962) or bybinding of tritiated cAMP and precipitation with ammonium sulphate asdescribed further below. Especially preferably the binding agent is achimeric fusion protein containing one or more cAPK B-sites, especiallyRIα B-sites, and a polypeptide region which is one member of a specificbinding partner pair (e.g. a biotin binding site of streptavidin) orwhich is a surface binding extension (e.g. Gly Gly Cys or GST(glutathione S-transferase)). Such chimeric fusion proteins comprisingdisabled cAPK A-sites and one or more cAPK B-sites may readily beprepared by conventional techniques, eg transfection of bacterial hostssuch as E. coli with plasmids incorporating in an operable reading framean appropriately coded nucleic acid sequence. In an especially preferredembodiment of the invention the binding agent comprises a B-sitemodified relative to a native mammalian cAPK B-site by replacement ofCys by Ala or by another amino acid residue that cannot form adisulphide bridge. This can be done by mutagenesis as discussed above inrelation to A-site disablement. Such binding agents containing aCys-free B-site are more stable on storage than binding agents with Cysresidues in the B-site.

The binding agent is preferably used in aqueous solution undernon-oxidizing conditions; typically the solution will contain one ormore chelating agents such as EDTA and EGTA as stabilizers, and areducing agent such as DTE, DTT, 2-mercaptoethanol or glutathione.

Where the assay method of the invention involves competition for bindingbetween the cAMP in the sample and a labelled cAMP, it is preferred thatthe label be tritium (³H) and/or that the label be attached to asubstituent at the 8-position, i.e. as a substituent on the adenosinering structure. cAMP 8-labelled with ¹²⁵I (generally attached via alinker group) is novel and forms a further aspect of the presentinvention.

³H-cAMP is available commercially from Amersham plc, UK.

The label in the labelled cAMP may be any species which is detectable(e.g. by virtue of radiation emission or absorption) or which cangenerate a detectable species or event (e.g. by virtue of enzymaticactivity). Radiolabels, chromophores and fluorophores are preferred asthe label may be placed so as not to interfere with or to minimallyinterfere with the ability of the labelled cAMP to bind to the cAMPbinding agent. In one embodiment of the invention the labelled cAMP issurface-bound cAMP with the conjugates formed by binding of the cAMPbinding agent to the surface-bound cAMP being detectable for example bysurface plasmon resonance. Such surface binding of cAMP may be effectedfor example by reacting a substrate having surface carboxyls oractivated carboxyls with an 8-aminoalkanoylamino-cAMP (e.g.8-aminooctanoyl amino-cAMP, available from BioLog, Bremen, Del.). Inthis embodiment the linker between the surface (e.g. the polymerbackbone of a polymeric substrate) and the pendant cAMP groupspreferably has a molecular weight of less than 1000D, more preferably amolecular weight of 100 to 500D. (In this way the SPR signal differenceon binding of the binding agent to the surface is optimised.) In anotherembodiment of the invention the labelled cAMP is a polymer (e.g. adendrimer) with pendant cAMP groups (preferably 8-attached). LabelledcAMP:binding agent conjugates can in this embodiment be detected bylight scattering (e.g. turbidimetry or nephelometry). Where the labelledcAMP is radiolabelled it is preferably ³H-cAMP (detectable byscintillation) or 8-(¹²⁵I-X)-cAMP (where X is an organic group providinga chain of up to 25 atoms to link the iodine to the 8-position, e.g. atyrosine-alkanoylamino group). Alternatively ³²P-labelled cAMP(available from ICN Pharmaceuticals) may be used. Preparation oflabelled cAMP can be effected by conventional chemical synthesistechniques. Thus for example 8-amino-octanoyl-amino cAMP (available fromBioLog, Bremen, Del.) may be reacted with tyrosine to introduce atyrosine residue at the 8-position. This can be iodinated with ¹²⁵Iusing a standard kit (e.g. operable to effect peroxidase catalysediodination using an inorganic ¹²⁵I compound). The bulky substitution ofthe 8-position does not appear to inhibit binding of the cAMP to a cAPKcAMP binding site.

In one especially preferred embodiment of the invention, conjugates ofcAMP and of labelled cAMP with the cAMP binding agent are precipitatedand taken up by a filter, e.g. a membrane filter. In this embodiment,the conjugate precipitation is conveniently effected using a sulphatesolution, e.g. aqueous ammonium sulphate. The cAMP binding sites of cAPKdo allow for cAMP exchange (i.e. complex dissociation) and so it isdesirable to “freeze” the quantity of labelled cAMP in the precipitate.This can readily be done by the use of a precipitation agent comprisinga solution of cAMP and sulphate. The solution is preferably almostsulphate saturated, e.g. 55-95% saturated, more especially 75-92%saturated and preferably contains cAMP in a quantity in excess of thelabelled cAMP in the sample to which it is to be added, eg at a 0.1 mMconcentration. In general this “stop” solution will be added cold in avolume ratio of about 20:1 to 1:2, more preferably about 15:1 to 5:1relative to the sample. Typically the stop solution is applied attemperatures below 10° C., e.g. 0° C., and if there is a delay betweenstop solution application and separation of the precipitated conjugatefrom the supernatant, the sample should be held cold during this delayperiod, e.g. at 0° C. The stop solution is preferably buffered, e.g. atpH7.

In this embodiment, following conjugate precipitation, the precipitateand supernatant are preferably separated by filtration, e.g. over amicrometer range pore size filter (e.g. 0.45 μm pore size). Suitablefilter membranes are available commercially from Millpore, e.g. HAWP ormore preferably HAMK filters.

After filtration, the filter membrane is preferably rinsed to removeuncomplexed labelled cAMP. This is preferably done immediately afterfiltration and is preferably repeated, e.g. one or two further rinses.Diluted stop solution may be used as the rinse solution; generallyhowever this need not contain cAMP.

Where the sample has a high salt content, filtration should be effectedas soon as possible after precipitation, e.g. within 30 minutes.

Where the sample under investigation has a low binding agentconcentration, it is desirable to add casein to the stop solution topromote precipitation. Where the sample under investigation has a highbinding agent concentration, it may be desirable to use glass fibrepre-filters.

Following precipitation and rinsing, the quantity of labelled cAMPretained by the filter membrane may be detected. In the case ofchromophore or fluorophore labelling or labelling with radiolabels otherthan those requiring scintillation counting, the signal may be read fromthe membrane or following release of the conjugate or the labelled cAMPfrom the membrane. Where detection involves scintillation counting, e.g.as with tritium labelled cAMP, the conjugate or the labelled cAMP mustbe released from the membrane. This may conveniently be effected bycontacting the membrane with an aqueous surfactant solution, e.g. asodium dodecyl sulphate solution, typically a 1-5% w/v solution,especially a 2% w/v solution. The conjugate or the labelled cAMPreleased into solution may then be detected in the solution, e.g. in thecase of ³H-cAMP by addition of a scintillation fluid (e.g. “Emulsifiersafe for aqueous samples” available from Packard).

As with most diagnostic assays, the assay system for use according tothe invention will generally require calibration against standards (i.e.cAMP solutions) with a range of known cAMP concentrations. The assay kitof the invention will thus typically be supplied with a set of standardsfor calibration and/or a calibration chart and/or a computer program ordataset for interpolation or extrapolation from signal values forstandards to determine the cAMP content of the sample underinvestigation.

The sample investigated using the method of the invention will typicallybe of a biological or biologically derived material, e.g.microorganisms, cells, tissues, body fluids, body organs, etc. Ofparticular interest are samples of or derived from microorganisms,especially bacteria and yeasts. Also of interest are samples of orderived from multicellular organisms, e.g. mammals, reptiles, birds andfish especially humans. It should be noted however that cAMP values willgenerally be lower in cell-free body samples than in body cells, e.g.about nanomolar levels in serum as opposed to micromolar levels inintracellular fluids.

In many biological samples, once removed from the host species the cAMPcontent reduces rapidly over time. For this reason, the samples used inthe assay method of the invention will generally be pretreated to reduceor eliminate cAMP degradation. Typically this may involve addition of abuffer and/or a chelating agent (e.g. EDTA) . For assays of body tissueor organ samples, since cAMP levels can drop very rapidly on sampleextraction or on the subject's death or exposure to stress, it may bedesirable to freeze the sample as quickly as possible after extraction,e.g. by plunging into liquid nitrogen. The sample may then be processedfurther, e.g. ground, solvent extracted, etc, before assay performance.

The method of the invention is a method of assaying for cAMP. This mayinvolve generation of a quantitative, semiquantitative or qualitativeresult, e.g. the concentration of cAMP in the sample, the concentrationof cAMP relative to another analyte, allocation of the result to a bandin a multi-band representation of cAMP concentration, indication of thecAMP concentration as being above (or below) a predetermined thresholdvalue (e.g. one indicative of a normality/abnormality boundary or amild/severe boundary), or even an indication of a predicted outcome,e.g. the period for which a whole blood sample may continue to be storedunder refrigeration while still being suitable for use in a transfusion.(For the latter, the sample tested will generally be lysed citratedwhole blood or red blood cell concentrate). All such determinations ofcAMP content are considered to fall within the assay method of theinvention.

Besides assaying for cAMP, the assay method of the invention may be usedto assay for other cyclic nucleotides (e.g. cGMP) and cyclic nucleotideanalogs that are capable of binding to the cAPK cAMP binding site. Ifsuch assays are competitive assays, then the competing labelled analytemay be a labelled cAMP or, more preferably, a labelled version of thedesired analyte. Such assays are of particular importance in theinvestigation of the biodistribution and pharmacokinetics of candidateor actual cyclic nucleotide (analog) drug compounds or precursors.Unlike the conventional cAMP assays referred to above, the assay methodof the invention is sensitive and accurate enough to detect suchcompounds. In such assays, if desired, the sample may be treated toreduce or eliminate any cAMP content which might interfere, e.g. bytreating the sample with an agent to which cAMP binds but to which thecandidate or drug does not or which serves to transform cAMP into aspecies which does not bind at the cAPK cAMP binding site, e.g. anenzyme with phosphodiesterase activity which serves to transform cAMP(but not the candidate or drug) into a species which does not bindsignificantly at the cAPK cAMP binding site, or an antibody which bindscAMP but not the candidate or drug.

Examples of cyclic nucleotides and analogs which can be assayed for inthis way include 8-aminohexylamino-cAMP, 8-bromo-cAMP, 8-chloro-cAMP,8-chlorophenylthio-cAMP, N6-monobutyryl-cAMP and cGMP.

Thus viewed from a further aspect the invention provides a method forassaying for a cyclic nucleotide or cyclic nucleotide analog, saidmethod comprising contacting said sample with a polypeptidic bindingagent capable of binding said cyclic nucleotide or cyclic nucleotideanalog and optionally also with a labelled competitor species capable ofbinding to said binding agent, and detecting conjugates of said bindingagent with said cyclic nucleotide or cyclic nucleotide analog or saidcompetitor species, characterized in that said binding agent comprisesfunctional cAPK cAMP B-binding sites only.

Viewed from a still further assay the invention also provides a kit foran assay for a cyclic nucleotide or cyclic nucleotide analog, said kitcomprising a polypeptidic primary binding agent capable of binding saidcyclic nucleotide or cyclic nucleotide analog; optionally a labelledcompetitor species capable of binding to said binding agent; andoptionally a secondary binding agent; characterized in that said primarybinding agent comprises functional cAPK cAMP B-binding sites only.

This kit and assay method will preferably utilize the features mentionedabove in connection with the cAMP assay and kit.

The assays of the invention, unlike the prior art antibody-based assays,are insensitive to the presence of divalent metal ions up to relativelyhigh concentrations (e.g. 10 mM). This is of importance since the labileconcentrations of such metal ions (e.g. Ca²⁺ and Mg²⁺) are stronglyaffected by the presence of the chelating agents which are required tostabilize cAMP concentrations in samples before the assays areperformed.

All publications referred to herein are hereby incorporated byreference.

The assay method of the invention will now be described further byreference to the following non-limiting Examples.

EXAMPLE 1 cAMP Binding Agent Preparation

The NcoI blunt end fragment of the cDNA for a fusion polypeptide of GSTand non-mutagenized human RIα containing a thrombin cleavage sitebetween GST and RIα was inserted in the NcoI-HindIII sites of plasmidpGEX-KG, the insert corresponding to nucleotides 103 to 1474 of thepublished sequence.

For mutagenesis a QuickChange Site-Directed Mutagenesis Kit (availablefrom Stratagene) was used. The RIα encoding double stranded plasmid andtwo synthetic oligonucleotide primers with the desired mutations wereannealed and extended by means of the Pfu DNA polymerase. The syntheticoligonucleotide primers were 5′-GGAGGGAGCTTTGAAGAACTTGCTTTG and3′-CCTCCCTCGAAACTTCTTGAACGAAAC. After temperature cycling, the parentalDNA template was digested using Dpn 1. The mutated DNA was transformedinto Epicurian Coli® XL1-Blue supercompetent cells. The mutations wereconfirmed by sequencing (ABI Prism 3700).

The plasmid was transformed into E. coli BL21, and preincubated for 2hours before expression was induced with 0.4 mM IPTG. The bacterialincubation was performed at 25° C.

The expressed GST hRIα fusion protein was purified by binding toglutathione-agarose (Pharmacia), according to the manufacturer'sprotocol. The fusion protein was further purified on a Superdex 200 gelfiltration column (Pharmacia FPLC system). The GST-tag was cleaved offby thrombin followed by an additional FPLC size exclusion chromatographystep.

SDS-PAGE mobility of GST-hRIα fusion protein corresponded to theexpected value of 81 kDa using bovine serum albumin, hen ovalbumin andBio-Rad high molecular weight standards for calibration.

The thrombin cleaved protein had SDS-PAGE mobility of 51 kDa.

EXAMPLE 2 Assay Performance

Sample Preparation

Male Wistar rats (120-400 g) were anaesthetized, the liver exposed, anda biopsy snap-frozen between the metal clamps of a Wollenberger tongprecooled in liquid nitrogen. The frozen tissue was pulverized. (Thepowder could be stored in liquid nitrogen for months without cAMPdegradation). To extract cAMP the powder was precipitated in ice-cold 5%(w/v) aqueous trichloroacetic acid in 0.1 M HCl (1 ml per 50 mg tissuepowder), and centrifuged (20,000×g av.) for 10 minutes. Thetrichloroacetic acid was removed from the supernatant by repeated (4×)extraction with at least 4 volumes of water-saturated diethyl ether. Thesample was neutralized by the addition of 20 μl of 10 M NaOH.

Assay Performance

100 μL of sample is mixed with 50 μL of 3 nM ³H-cAMP (Amersham plc, UK)and then with 50 μL of the thrombin cleaved binding agent of Example 1(1.2 nM in cAMP binding sites) in 50 mM HEPES (adjusted to pH 7.4 withdipotassium phosphate and containing 20 mM EDTA, 3 mM EGTA, 0.5 mg/mLserum albumin, 0.2 mg/mL soybean trypsin inhibitor and 0.5 mM DTE). Themixture is incubated for 2 to 18 hours and then mixed vigorously with 1mL ice cold 80% saturated ammonium sulphate.

2×2 mL of ice cold 65% saturated ammonium sulphate is passed through a25 mm diameter membrane filter (0.45 μm pore size, e.g. HAMK availablefrom Millipore) whereafter the precipitated sample is passed through thesame filter under suction. Immediately after the sample has run through,2 mL of ice cold 65% saturated ammonium sulphate are applied followed bya further two rinses with 2 mL of ice cold 65% saturated ammoniumsulphate.

The filter is transferred to a scintillation vial containing 1 mL of 2%w/v aqueous sodium dodecyl sulphate solution. The precipitate isdissolved by vortexing for 10 minutes. Thereafter 7 mL of scintillationfluid (“Emulsifier Safe for aqueous samples” from Packard) are added andthe vial is counted in a beta-counter for at least 4 minutes.

A calibration curve, prepared using the same assay procedure andstandard solutions of cAMP in 50 mM HEPES (adjusted to pH 7.4 withdipotassium phosphate and containing 20 mM EDTA, 3 mM EGTA, 0.5 mg/mLserum albumin, 0.2 mg/mL soybean trypsin inhibitor and 0.5 mM DTE) isused to determine the cAMP content of the sample.

EXAMPLE 3 Assay Performance

Where the sample contains a low quantity of cAMP or a high concentration(e.g. 10 mM) of a weakly binding substance (e.g. ATP or AMP) which candissociate from the binding agent post-incubation, the assay of Example2 may be performed omitting the ³H-cAMP from the initial incubation andsubsequently performing a one hour post incubation in the presence of 10nM ³H-cAMP. The procedure is otherwise unchanged.

1. A method for assaying for cAMP in a sample, said method comprisingcontacting a sample with an unknown cAMP content with a polypeptidiccAMP binding agent and optionally with a labelled cAMP and detectingconjugates of cAMP or labelled cAMP and said binding agent,characterized in that said binding agent comprises functional cAPK cAMPB-binding sites only.
 2. A method as claimed in claim 1, wherein saidbinding agent has disabled A-binding sites.
 3. A method as claimed inclaim 1, wherein said binding site is an RIα B-site.
 4. A method asclaimed in claim 1, wherein said labelled cAMP is labelled at the8-position by iodine-125.
 5. A method as claimed in claim 1, whereinsaid labelled cAMP is attached to a substrate surface at the 8-position.6. A method as claimed in claim 1, wherein said cAMP conjugates aredetected using surface plasmon resonance.
 7. A method as claimed inclaim 1, wherein said labelled cAMP is labelled with tritium.
 8. Amethod as claimed in claim 1, wherein said binding site is capable ofbinding cAMP with a K_(D) of less than 300% of that of the site innative human cAPK.
 9. A method as claimed in claim 1, wherein saidbinding site is capable of binding cAMP with a K_(D) of less than 110%of that of the site in native human cAPK.
 10. A kit for a cAMP assay,said kit comprising a polypeptidic primary binding agent capable ofbinding cAMP; optionally, a labelled cAMP; and optionally a secondarybinding agent; characterized in that said primary binding agentcomprises functional cAPK cAMP B-binding sites only.
 11. A polypeptidiccAMP binding agent which comprises functional cAPK cAMP B-binding sitesonly, and compositions and items comprising said binding agent.
 12. cAMPlabelled at the 8-position by iodine-125, and compositions thereof.13-14. (canceled)
 15. A method for assaying for a cyclic nucleotide orcyclic nucleotide analog, said method comprising contacting said samplewith a polypeptidic binding agent capable of binding said cyclicnucleotide or cyclic nucleotide analog and optionally also with alabelled competitor species capable of binding to said binding agent,and detecting conjugates of said binding agent with said cyclicnucleotide or cyclic nucleotide analog or said competitor species,characterized in that said binding agent comprises functional cAPK cAMPB-binding sites only.
 16. A kit for an assay for a cyclic nucleotide orcyclic nucleotide analog, said kit comprising a polypeptidic primarybinding agent capable of binding said cyclic nucleotide or cyclicnucleotide analog; optionally, a labelled competitor species capable ofbinding to said binding agent; and optionally a secondary binding agent;characterized in that said primary binding agent comprises functionalcAPK cAMP B-binding sites only.